The glycoprotein E (gE) of pseudorabies virus (PRV) is known to be an important marker protein in the control and eradication of Aujeszky's disease. In this study, BALB/c mice were immunized with gE-deleted PRV as tolerogen and with wild-type PRV as immunogen. The spleen cells from the immunized mice were then fused with the myeloma cell line Sp2/0. Two hybridoma cell lines that could stably secrete the monoclonal antibody (MAb) against gE were achieved by using indirect ELISA screening and subcloning three times; they were named 1D2 and 2B2. Indirect immunofluorescence assay (IFA) revealed that the MAbs were specifically against gE of PRV. MAbs 1D2 and 2B2 were subgroup IgG1. The MAbs obtained in this study provide useful tools for the development of differential diagnostic methods for PRV.